ZP2 and ZP3 co-localize in the endoplasmic reticulum and in 1-5 um post-Golgi structures comprised of multivesicular aggregates (MVA), but a co-immunoprecipitation assay does not detect physical interactions. In addition, ZP2 trafficks normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N-glycosylation has been implicated for correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, EGFP-tagged ZP3 lacking N-glycosylation sites is present in MVA and is incorporated into the zona matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffick independently through the oocyte prior to assembly into the extracellular zona pellucida.[unreadable] [unreadable] The molecular basis of taxon-specific sperm-egg recognition remains to be determined. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida is composed of ZP1, ZP2 and ZP3 which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raised the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3 and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.